UNIVERSITY OF MEDICINE AND

PHARMACY OF CRAIOVA

DOCTORAL SCHOOL

PHD THESIS

ABSTRAT

CLINICAL, HISTOLOGICAL AND

IMMUNOHISTOCHEMICAL STUDY

IN ENDOMETRIOSIS

PHD COORDINATOR,

PROFESSOR MOGOANTĂ LAURENŢIU

PHD STUDENT,

OFIŢERU (ISTRATE-OFIŢERU) ANCA-MARIA

CRAIOVA

2019

CONTENT

GENERAL PART

INTRODUCTION..........................................................................................3

I. ONTOGENY OF THE FEMALE REPRODUCTIVE TRACT............5

II. HISTOPHYSIOLOGY OF THE UTERUS............................................6

III. ENDOMETRIOSIS.................................................................................9

SPECIAL PART

IV. OBJECTIVES........................................................................................12

V. CLINICAL-STATISTICAL STUDY OF ENDOMETRIOSIS..........12

VI. HISTOLOGICAL STUDY OF ENDOMETRIOSIS.........................16

VII. IMMUNOHISTOCHEMICAL STUDY IN ENDOMETRIOSIS...18

VIII. FINAL CONCLUSIONS...................................................................23

SELECTIVE BIBLIOGRAPHY...................................................26

3

GENERAL PART

Introduction

Endometriosis is a fairly common benign pathology that affects

most women and can have multiple locations: pelvis, peritoneum,

rectovaginal septum, ovaries, abdominal wall after cesarean section,

umbilicus and more less commonly in other organs: kidney, urinary tract,

colon, lungs or even brain, and also, the eutopic location, in the myometrium

structure, in which case it is called adenomyosis [Shalin, S.C., Haws, A.L., şi

colab., 2012; Stevens, E.E., Pradhan, T.S., şi colab., 2013; Efremidou, E.I.,

Kouklakis, G., şi colab., 2012; Brătilă, E., Ionescu, O.M., şi colab., 2016].

The most common symptoms are pelvic pain, vaginal bleeding,

infertility, dysmenorrhea, dysuria, dyspareunia. The severity and complexity

of the pain involve major problems in managing the therapeutic possibilities

[Laux-Biehlmann, A., D’Hooghe, T., şi colab., 2015; McKinnon BD,

Bertschi D, şi colab., 2015].

The etiopathogenesis of this condition can be divided into five

categories: the most accepted theory being that of retrograde menstruation,

but also the coelomic metaplasia, the origin of the remaining embryonic cells,

the theory of induction and the lymphatic or vascular dissemination are

mentioned [Berceanu, C., Ofițeru, A.M., şi colab., 2018].

It should be kept in mind that in addition to the symptomatology that

may develop unfavorably, there may be associated risk factors that alter the

structure of the endometrial glands, leading to premalignant or even

malignant transformation of endometriosis outbreaks.

Studies have shown that genes, the immune system, environmental

factors and hormonal influence can lead to endometrial hyperplasia, dysplasia

and even malignant transformation of ectopic endometriosis outbreaks

[Berceanu , C., Ofițeru, A.M., şi colab., 2018].

4

From a microscopic point of view, this pathology is characterized by

the presence of the endometrial glands and the endometrial stroma,

accompanied by different degrees of fibrosis, old or recent hemorrhage and

macrophages that have phagocytized hemosiderin [Comănescu, M., Potecă,

A., şi colab., 2018].

The epithelial component may have several aspects, may be

unistratified, müllerian type or may have glands with different architectures,

but which respect the characteristics of the normal endometrium

[Comănescu, M., Potecă, A., şi colab., 2018].

The stroma is represented by round ovary cells with spherical nuclei

and reduced cytoplasm. Peristromal cells of the inflammatory system, T

lymphocytes, rare B lymphocytes and numerous macrophages that have

phagocytised hemosiderin are identified. This inflammatory

microenvironment is associated with endothelial dysfunction and participates

in the carcinogenetic transformation of the endometriosis islands

[Comănescu, M., Potecă, A., şi colab., 2018].

All these microscopic changes occurring in endometriosis /

adenomyosis outbreaks, together with the symptomatology, clinical signs and

possible tissue transformations, place this topic in a current field of research,

based on interdisciplinary studies.

KEYWORDS: endometriosis, adenomyosis, symptomatology,

microscopy.

5

CHAPTER I. Ontogeny of the female reproductive tract

Although the chromosomal sex or genetic sex of an embryo is

determined at the time of fertilization by the type of sperm that fertilizes the

egg, the male and female morphological characteristics begin to develop only

in the seventh week of pregnancy. The early genital systems of the two sexes

are similar; therefore, the initial period of genital development is referred to

as the indifferent state of sexual development [Moore, K.L. Persaud, T.V.N.,

2003].

Female gonads, ovaries, derive from three sources: the mesothelium

(mesodermal epithelium) lining the posterior abdominal wall, the underlying

mesenchyme (embryological connective tissue) and from the primordial germ

cells [Moore, K.L. Persaud, T.V.N., 2003].

Both sexes initially show two pairs of genital ducts: the mesonephric

duct or Wolffian and the paramesonephric duct or Müllerian. The second

appears as a longitudinal invagination of the surface epithelium of the antero-

lateral area of the urogenital ridge.

Initially, the two ducts are separated by a septum, later merging and

giving rise to the uterine canal. The lower extremity of these two associated

ducts is projected into the urogenital sinus, at the level of the posterior wall,

where it forms a small protrusion, called the paramezonephric tubercle

(Műller) [Sadler, T. W., 2007].

The SRY gene, via the SOX9 gene, participates in the development of

male gonads, and the WNT gene induces ovarian development. WNT

amplifies DAX1 expression belonging to the family of genes encoding

nuclear hormone receptors, with the role in inhibiting SOX9 action. Also,

WNT4 regulates the expression of genes responsible for the differentiation of

female gonads - the ovaries, including the TAFII105 gene that belongs to the

RNA polymerase of ovarian follicular cells [Sadler, T.W., 2007].

The main genital tract arises from the paramezonephric ducts in the

female embryos. First, each duct is composed of 3 portions: 1) a vertical

6

upper segment, communicating with the abdominal cavity; 2) a segment

placed horizontally that intersects with the mesonephric duct; 3) a lower

vertical segment which merges with the opposite homologous segment. The

first two enumerated (listed) segments will form the uterine tube, after the

ovary descends, and the lower merged segments give rise to the uterine canal

[Sadler, T.W., 2007].

After the fused extremity of the paramezonephric ducts reaches the

urogenital sinus, two compact projections, the sinovaginal bulbs, begin to

form in the pelvic portion, which will form a compact vaginal plate [Sadler,

T.W., 2007].

In conclusion, the urinary and genital apparatus (systems) derive from

the mesoderm. The 3 components of the genital system: the gonads, the

genital tract and the external genital organs go through the indifferent state.

Under the control of the WNT4 gene, the ovaries will develop, amplifying

the expression of the DAX1 gene, leading to the inhibition of the SOX9 gene

(involved in the development of male genital systems). Estrogens act on the

paramezonefric system in females, directing the formation of the uterine

tubes, uterus, cervix and upper portion of the vagina [Sadler, T.W., 2007].

CHAPTER II. Histophysiology of the uterus

The female genital tract is composed of female gonads (ovaries), with

gametogenic and hormonogenic functions, female genital tract: Fallopian

tubes, uterine and vaginal tracts, from external genital organs:labia majora,

labia minora, clitoris and vaginal vestibule, but also from hormone-dependent

structures such as: mammary gland and placenta.

The uterus is a pear-shaped organ, divided into: uterine body, isthmus

and cervix; located between the bladder and rectum, superior to the vagina,

normally in an anteverted and anteflexed position [Crăițoiu, Ş., 2003].

7

The uterine wall has 3 tunics: the perimeter (outer layer),

myometrium (middle tunic) and endometrium (internal tunic). [Crăițoiu, Ş.,

2003].

The uterine mucosa is composed of an epithelium and a chorion. In

the fertile period of the woman (between puberty and menopause), the

endometrium is made up of an epithelium and a lamina propria, which

contains straight tubular glands. In the portion near the myometrium these

glands sometimes have a branched appearance. The endometrial epithelium is

located on a basement membrane with a continuous structure, which

separates it from the cytogenetic chorion and presents a single row of few,

secretory, ciliated cells, with short and scattered kinocilia and basal,

proliferative, regenerating cells [Crăițoiu, Ş., 2003].

The epithelium of the endometrial glands resembles that of the

superficial endometrium, but has fewer ciliated cells [Junqueira L.C.,

Carneiro, J., 2008].

Female hormones, estrogen and progesterone, control the activity of

the female reproductive tract. Epithelial cells and associated connective tissue

are proliferating and differentiating under hormonal control. Starting with

puberty the pituitary hormones (follicle-stimulating-FSH and luteotropic-

LTH) are triggering the ovarian secretion of estrogen and progesterone,

which induce endometrial cyclic changes [Junqueira L.C., Carneiro, J.,

2008].

The menstrual phase lasts for a few days, on average 3-4 and is

followed by the proliferative phase and the secretory (luteal) phase. The

proliferative phase varies with duration, averaging 10 days, and the secretory

phase starts with ovulation and lasts 14 days (Table 1) [Junqueira L.C.,

Carneiro, J., 2008].

The uterine arteries, direct branches of the common iliac arteries, are

the largest (main) blood supply to the uterus. The ovarian arteries and the

round ligament arteries supply the uterus with a smaller quantity of blood.

8

From the uterine arteries emerge the arcuate arteries that branch in radial

arteries, which send collaterals to the cervix and the uterine body. From the

uterine arterial branches that penetrate into the myometer will emerge

branches that form the plexiform layer, a true arterial anastomotic plexus; the

arteries that supply the 3 layers of the uterus arise from this plexus.

Two types of arteries supply the endometrium: short, straight,

nutritious and long, functional, spiral. The long, functional, hormone-

dependent, spiral arteries, also called helicine arterioles (spiral arteries),

supply the superficial layers of the endometrium. During the proliferative

phase, they increase in size, capillaryize massively, form arterial-arterial

anastomoses in the 2/3 endometrial surfaces and twist strongly during the

secretory phase of the endometrial cycle. At the end of the progesterone

phase, the wall of the functional arteries is strongly contracted due to the

sudden decrease of the ovarian hormones and a process of local ischemia is

triggered, followed by endometrial necrosis.

The venous blood flows in the opposite direction compared to arterial

blood. And the veins form numerous venous plexuses in the myometrium that

are better represented in pregnancy.

Lymphatic vessels have a particular importance in uterine malignant

pathology. Each tunic of the uterus has an intrinsic lymphatic network:

mucous, muscular and serous. These networks lead to the formation of

collecting trunks which drain near the uterine margins and to the groin and

iliac ganglia.

The uterine innervation is especially vegetative, parasympathetic and

sympathetic, receiving branches from the inferior hypogastric plexus [Bold,

A., Mogoanta, L., şi colab, 2011].

9

CHAPTER III. Endometriosis

Endometriosis is a truly enigmatic disease, various hypothesis have

been studied for nearly a century without being fully understood.

The histogenesis of this pathology is not known, but the obligatory

feature in the definition of endometriosis is the presence of endometrial tissue

in locations outside the uterine cavity or in the myometrium structure, also

known as adenomyosis.

Morphologically, endometriosis and adenomyosis are represented by

the existence of endometrial tissue and periglandular stroma in an ectopic

region, but, from the anatomical and clinical point of view, the two

pathologies are different. There are several theories regarding the

pathogenesis of this condition (Table 1).

Phases of the menstrual cycle

Follicular

phase

(Proliferative)

Luteal phase

(Secretory)

Menstrual

phase

Pituitary

hormones

and their

main

actions

FSH (follicle-

stimulating

hormone causes

accelerated

growth of

ovarian

follicles

LH (luteinizing hormone) reaches a

peak that coincides with the onset of

the secretory phase and occurs as a

result of estrogen-stimulating action.

It induces ovulation and the evolution

of the yellow body

Events

occuring in

ovaries

Pre-antral and

antral ovarian

follicles. The

dominant

follicle reaches

the

preovulatory

stage

Ovulation

The

evolution

of the

yellow

body

(corpus

luteum)

The

involution of

the yellow

body (corpus

luteum)

10

Ovarian

hormones

Ovarian

follicles secrete

estrogen that

influences (acts

on) the uterus,

vagina and

fallopian tubes

The progesterone

secreted by the

yellow body has a

main action on the

uterus

The

production of

progesterone

is stopped

Main

endometrial

events

Regeneration of

the mucosa

after

menstruation

Thickening of the mucosa, spiraling

of the glands, occurrence of glandular

secretion

Elimination of

the superficial

2/3 of the

endometrium,

approximately

14 days after

ovulation

Table 1 – Events that occur during the menstrual cycle - synthesis.

Current theories Multifactorial etiology Risk factors

Retrograde

menstruation or

transtubar

regurgitation, tissue

transplantation

INTERACTION

Specific genes

Environmental factors

Family aggregation

Lymphatic

dissemination

Genetic mutation and

polymorphism

Hematogen / vascular

dissemination

Anatomical defects

Metaplasia of the

celomic epithelium

Toxins in the

environment

Induction theory

Hormonal dependence

Immunological factors

/ The role of the

immune system

Tabelul 2 - Table 2 - Theories regarding the pathogenesis of

endometriosis) [after Berceanu, C., Brătilă, E., şi colab. 2018].

11

Depending on the location of endometriosis, the associated risk

factors various malignant transformations of this pathology have been studied

(Figure 1).

Figure 1 - Malignant tumors arising from endometriosis (endometriotic

transformation).

12

SPECIAL PART

CHAPTER IV. Objectives

The purpose of this study is to evaluate and statistically analyze a

group of patients with endometriosis/ adenomyosis in the purpose of fully

grasping its clinical characteristics (symptomatology, personal antecedents

both physiological and pathological, behavioral factors, laboratory analysis),

diagnosis methods and the applicable treatments.

Also, another goal is to highlight certain histological,

histopathological and immunohistochemical aspects of this pathology,

depending on localization (ovarian, pelvic/ peritoneal, myometrial, parietal)

and evaluating the endometrial focal abnormalities/ ectopic glandular

abnormalities, in order to demonstrate the involvement of some risk factors in

the evolution of this pathology.

Through immunohistochemistry, aided by specific markers of this

pathology (cytokeratin 7, estrogen receptors and progesterone receptors) I

wanted to highlight the presence of endometriosis/ adenomyosis in the

structure of different organs and to establish a positive diagnosis regarding

the tissue origin. With the aid of the differential marker (cytokeratin 20) i

wanted to show eventual distinctive areas containing metastatic ectopic

glandular proliferation with digestive origin.

Through numerical analysis, with the help of required

immunohistochemical markers (cluster of cluster of differentiation 31/34,

cluster of cluster of differentiation 3/20/68/79α, tryptase, proliferation marker

Ki67, tumor protein p53, regulator protein BCL-2 and PTEN) I aim to

underline the involvement of increased peri-endometrial vascularization,

inflammation, cell proliferation factors, the presence of regulator protein and

13

tumor suppressor protein, in the development of endometriosis/ adenomyosis

and their eventual preneoplastic/neoplastic conversions.

CHAPTER V. Clinical-statistical study of endometriosis

Introduction: Endometriosis is a benign pathology that affects

especially women of reproductive age. The most common symptoms

encountered in this pathology are pain, subfebrility, bleeding, but also

dysmenorrhea, dysuria, dyspareunia. The severity and complexity of the pain

can cause major problems in the management of therapeutic resources.

[Laux-Biehlmann, A., D’Hooghe, T., şi colab., 2015; McKinnon, B.D.,

Bertschi, D., şi colab., 2015].

Medication is the first-line therapy [Walch, K., Unfried, G., şi colab.,

2009], but the absence of a favorable response may resort to surgical

treatment. The first intention in surgical treatment is laparoscopy. Depending

on the location of the endometriosis outbreaks and the depth of the lesion, the

surgical technique is chosen, which may represent the curative treatment of

this pathology [Crosignani, P.G., Vercellini, P., şi colab., 1996].

Material and methods: My study was carried out on 120 cases of

endometriosis, 30 cases of ovarian endometriosis, 30 cases of pelvic

endometriosis, 30 cases of adenomyosis (myometrial endometriosis) and 30

cases of abdominal wall endometriosis.

Patients were admitted and investigated in the Obstetrics-Gynecology

Clinic II and the Surgery Clinic III of the Craiova County Emergency

Clinical Hospital (SCJUC), between 2010-2018.

A clinical study and statistical analysis was performed using the

Microsoft Excel 2010 program, based on: the year of hospitalization,

localization of endometriosis (ovarian, pelvic, adenomyosis, endometriosis of

the abdominal wall), age of patients, environment of origin, height, weight,

body mass index ( BMI), behaviors (consumption of coffee, alcohol,

tobacco), living and working conditions (housewives / employees / student-

14

students), by symptomatology (bleeding, pain), personal pathological

antecedents, personal physiological background: menarche, regular or

irregular menstruation, spontaneous / on-demand abortions, number of

natural or caesarian section deliveries, infertility, surgery, clinical laboratory

data (leukocyte count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular

volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular

hemoglobin concentration (MCHC), platelet count, erythrocyte sedimentation

rate (ESR), serum value of Cancer Antigen 125 (CA-125 marker) and the

medication used to alleviate symptomatology, through which all patient data

were collected and compared.

Following the laboratory tests and the medical investigations, surgical

intervention was carried for the excision of the endometriosis outbreaks. The

surgical techniques were chosen according to the location of the

endometriosis.

Results: Age at diagnosis of endometriosis varied according to

location as follows: ovarian endometriosis (22-46 years old), pelvis (30-57

years old), adenomyosis (30-46 years), abdominal wall endometriosis (23-38

years old). Endometriosis of the abdominal wall occurs at younger age due to

the increase of births by caesarean section.

According to the year of diagnosis of endometriosis, it was observed

that most cases of ovarian endometriosis were in 2018, pelvic endometriosis

in 2017, adenomyosis in 2012 and 2017, and endometriosis of the abdominal

wall in 2017 and 2018.

Using the height and weight of each patient with endometriosis, we

calculated the body mass index and observed that most normal weight

patients had abdominal wall endometriosis (21 cases) and pelvic

endometriosis (21 cases), followed by adenomyosis (20 cases) and the fewest

normal-weight patients had ovarian endometriosis (16 cases). Underweight

patients-9 with ovarian endometriosis, 8 patients with abdominal wall

endometriosis and 4 patients with pelvic endometriosis. Overweight patients-

15

6 patients with adenomyosis, 2 patients with pelvic endometriosis and 2

patients with ovarian endometriosis. Class I obesity- a number of 4 patients

with adenomyosis and 2 patients with ovarian and pelvic endometriosis.

Grade II obesity- 1 patients with endometriosis of the abdominal, 1 patient

with ovarian e3ndometriosis and 1 patient with pelvic endometriosis.

The most common symptoms related to hospitalization were: vaginal

bleeding and pelvic pain.

Also according to the anatomical location, we compared the personal

physiological history (the presence of regular / irregular menstruation,

spontaneous / on-demand abortions, the number of eutocic births / by

caesarean section) and the personal pathological background (infertility,

surgery for the removal of endometriosis outbreaks: partial ovarectomy,

hysterectomy, excision of endometriotic nodules from the post-caesarean

scars), (Figure 2).

Studying the CBC (complete blood count), the erythrocyte

sedimentation rate, CA-125 marker, I also noticed that there were changes in

several cases. Behaviors of patients varied, being associated factors with the

development of endometriosis.

Figure 2 - Personal physiological and pathological history of patients with

endometriosis

16

Conclusions: The extensive symptomatology associated with

endometriosis can affect the physical as well as the mental state of the

patient. Dysmenorrhea, chronic pelvic pain and infertility are most

commonly associated with this benign pathology, which has the

characteristics of malignancy: it spreads locally and remotely, damages

adjacent tissues and causes cell invasion.

The treatment of endometriosis can be pharmaceutical, or, the second

therapeutic option, surgical, the technique being chosen according to the

location of the endometriosis and the status of the patient.

In the presence of risk factors: mechanical, chemical, genetic or

inflammatory, endometriosis outbreaks can turn premalignant or even

malignant.

The location of endometriosis varied according to the age of the

patients, the youngest had ovarian or abdominal wall endometriosis, which

appeared after caesarean section, and the oldest with pelvic endometriosis.

It has been studied that hormonal secretion is heavily influenced in

overweight women and it can disrupt menstruation, changing its flow and

implicitly rising the risk of endometriosis. In some women there is a directly

proportional relationship between the two, and in other women an inversely

proportional relationship.

CA-125 and ESR (1 hour) may be increased in patients with

endometriosis and can be used as screening markers for this pathology.

The inversely proportional relationship between smoking and

endometriosis was found by the interaction between cigarette smoke and the

glutathione-S-transferase gene polymorphism as a possible risk factor for the

development of endometriosis. It has been observed that patients consuming

alcohol or coffee may be more frequently affected by this pathology,

compared to women who do not consume these drinks.

17

CHAPTER VI. Histological study of endometriosis

Introduction: The positive diagnosis of endometriosis is supported

by the histopathological examination. Through the use of classical

histological techniques, through Hematoxylin-Eosin (HE) staining and

Masson's Trichrome staining (TRI), we have highlighted the presence of

eutopic / ectopic endometrial tissue.

From a microscopic point of view, endometriosis is a pathology

characterized by the presence of the endometrial glands and the endometrial

stroma, accompanied by different degrees of fibrosis, old or recent

hemorrhage and macrophages that have phagocytosed hemosiderin.

[Comănescu, M., Potecă, A, 2018].

Atypical endometriosis is characterized by the presence of cells with

pleomorphism that vary from moderate to severe, arranged in several layers

that can even form micro-papillae. This can potentially transform

premalignant, due to cellular atypia and architectural changes [Comănescu,

M., Potecă, A, 2018].

Malignant transformed endometriosis can be highlighted by

identifying endometriotic outbreaks with malignant transformation. The

diagnosis of malignancy associated with endometriosis pathology requires

the presence of the same tissue structures of the benign lesion, confirmation

of the primary origin and the histological continuity of the benign-malignant

lesions.

Material and methods: Following surgical treatment for solving

ovarian, pelvic (peritoneal) endometriosis, adenomyosis or endometriosis of

the abdominal wall, tissue of interest was harvested (collected), the tissue

samples were introduced in 10% neutral formalin and then embedded in

paraffin blocks. The blocks thus obtained were sectioned using the HM350

microtome and the resulting slides were stained using the classical

histological techniques Hematoxylin-Eosin and Masson’s Trichrome

18

stainings, all of which were performed within the Histology department of

the University of Medicine and Pharmacy of Craiova.

Conclusions: The diagnosis of endometriosis is dictated by the

histopathological examination. The classic HE and TRI stains identify the

glandular structures and the adjacent stroma with ectopic localization,

internally: adenomyosis or externally: in the structure of the pelvic or

abdominal organs. This histopathological diagnosis must sometimes be

confirmed by IHC.

The identification of endometriosis is based on meeting the following

criteria: the presence of the endometrial glands, the stroma and possibly the

perilesional hemorrhagic effusion, which contains macrophages that have

phagocytosed hemosiderin.

The epithelial component is represented by an epithelium that varies

in form and structure from simple columnar, to pseudistratified columnar or

pluristratified in hyperplastic premalignant transformations.

The stromal component is represented by young cells: fibroblasts,

lymphocytes and macrophages, involved in the triggering of inflammatory

processes and the eventual malignant transformation of endometriosis

outbreaks.

The diagnosis of malignant transformed endometriosis requires

immunohistochemical study and the fulfillment of obligatory

histopathological criteria: the coexistence of both lesions in the structure of

the same organ, with tissue continuity, but also the negation of the existence

of another neoplastic lesion, in order to eliminate the possibility of confusion

with a secondary determination.

CHAPTER VII. Immunohistochemical study in endometriosis

Introduction: Endometriosis can be identified using histological

staining, but a better differential diagnosis can be made by

19

immunohistochemistry techniques. Therefore, the 120 cases studied in this

thesis were also analyzed from an immunohistochemical point of view.

Using the anti-cytokeratin 7/20 antibodies (CK7, CK20), anti-

Estrogen (ER) / Progesterone (PR) receptors we have demonstrated that the

tissue areas we studied had endometrial origin.

Environmental, hormonal, inflammatory factors can influence these

areas, so that the presence of ER / PR can be altered, the degree of cell

proliferation may be increased (marking with anti-Ki67 antibody), the genetic

structures of B-cell lymphoma 2 (BCL- 2+) and Phosphatase and tensin

homolog (PTEN +) can be modified, tumor protein 53 (p53) may be positive

in atypical cases, inflammatory cell density may be increased compared to

the area adjacent to the normal endometrium: Cluster of differentiation

3/20/68 / 79α (CD3 +, CD20 +, CD68 +, CD79α +) and Triptase +, all of

which may influence cell structure, histo-architecture of the surrounding

microenvironment and cause premalignant or even malignant changes in

endometriosis outbreaks.

Immunohistochemical tests confirm the histological suspicion of

endometriosis. [Istrate-Ofiţeru, A.M., Pirici, D., şi colab., 2018].

Material and methods: The tissue sections were obtained similar to

those for the histological study and went through a process of antigenic

retrieval, blocking of endogenous peroxidase and blocking of non-specific

binding sites, then the sections were incubated with the primary antibody for

18 hours at 4 ° C, and the next day the signal was amplified by using a

peroxidase polymer system for 30 minutes (Nikirei-Bioscience, Tokyo,

Japan). The signal was then detected with 3,3'-diaminobenzidine (DAB)

(Dako, Glostrup, Denmark) and the slides were coated in a xylene-based

mounting medium (DPX, Sigma-Aldrich, St. Louis, MO, USA), after staining

with hematoxylin.

The microscopic images were photographed using a Nikon Eclipse

55i microscope, equipped with a 5 Mp color CCD camera, and analyzed with

20

the Image ProPlus 7 AMS software package (Media Cybernetics Inc.,

Buckinghamshire, UK).

For the proposed study, the sections were photographed in the regions

of interest, with the objectives 100 ×, 200 ×, and for quantification, 4 images

were produced with the objective 200 × for each case. The blood vessels and

cells of the inflammatory system were counted for each image separately and

then an average of the vascular / cellular density of the case was achieved. All

values were graphically represented and interpreted using Microsoft Excel

2013. The ANOVA test (ANOVA - variation analysis) was used for multiple

comparisons. In all cases, p <0.05 was used to indicate statistical

significance.

Results: With the help of immunohistochemical reactions, we

demonstrated that from a microscopic point of view the ectopic tissue ,post-

interventional, is endometrial.

With the help of the anti-CK7 antibody we showed that the areas of

interest have positive glandular epithelium, and to make the differential

diagnosis with a possible metastasis with digestive starting point, we

performed the immunolabeling with the anti-CK20 antibody, which reacted

negatively and showed that the tissue is not of digestive glandular type.

ER and PR are present in the endometrial cells, and the positive

reaction to the immunolabeling once again demonstrates the endometrial

origin.

For the study of peristromal vascularization we used the anti-CD34

antibody. The increased vascular density is observed periodically, especially

in the hyperplastic transformed areas. For the numerical quantification of

blood vessels, we took 4 photographs, with objective × 200 for each case, of

the lesion areas - periglandular (at the same distance from the endometrial

glands), depending on each location of the endometriosis and we observed

that the average density the highest number was present in the case of

abdominal wall endometriosis (23.69 CD34 + / × 200 vessels), followed by

21

the normal secretory-phase endometrium (26.06 CD34 + / × 200 vessels) and

the normal, eutopic endometrium proliferative (23.69 CD34 + / × 200

vessels), adenomyosis (18.6 CD34 + / × 200 vessels), peritoneal / pelvic

endometriosis (4.2 CD34 + / × 200 vessels) and ovarian endometriosis (2.7

CD34 + / × 200 vessels). ).

To study the cell proliferation rate we used the anti-Ki67 antibody. We

noticed that the marking for the cells going through cellular division is more

pronounced in the areas with hyperplastic transformation, compared to the

normal endometrium or endometriosis with different locations, but without

hyperplastic transformation.

For the study of preneoplastic or neoplastic cell transformations we

used the anti-p53 antibody to detect the cellular expression of a tumor

suppressor protein and we observed that it is more intensely expressed in

hyperplastic transformed than it is expressed in endometriotic lesions in cases

with ovarian or abdominal wall endometriosis.

Also, using the anti-BCL-2 antibody, we observed the expression of a

modified protein, which may have a regulatory role on programmed cell

death, in cells that can potentially go through malignant transformation. The

reaction is more intense if more genetic changes have occurred, especially in

cases with hyperplastic transformation, but also in those with ovarian or

abdominal wall endometriosis.

Similar to the anti-p53 antibody, the anti-PTEN antibody highlights

altered cells, in which a tumor suppressor gene was activated, which is

involved in regulating the cell cycle, preventing growth and over-accelerating

cell division. We observed that in cases with hyperplastic transformed

endometriosis, the reaction was more intense compared with the normal

endometrium or with the ectopic lesions, but without structural changes.

Given that the inflammatory process may be involved in cellular

structural and functional changes, we used several antibodies to determine

the number of inflammatory cells around endometriosis outbreaks with

22

different locations and those adjacent to the normal endometrium, so we

used: anti-CD3 antibody for T lymphocyte marking, anti-CD20 antibody for

lymphocyte B marking, anti-CD68 antibody for macrophage marking, anti-

Triptase antibody for mast cell marking, and anti-CD79α antibody all for

marking B lymphocytes.

We compared the results and observed that the highest average CD3 +

T-type lymphocyte density was found in the case of abdominal wall

endometriosis (116.19 cells / × 200), followed by adenomyosis (66.27 cells /

× 200). ), secretory phase eutopic endometrium (16.01 cells / × 200),

proliferative phase eutopic endometrium (13.56 cells / × 200), peritoneal /

pelvic endometriosis (12.93 cells / × 200) and ovarian endometriosis (12.82

cells / × 200).

The average CD20 + type B lymphocyte density varied as follows:

abdominal wall endometriosis (34.2 cells / × 200), adenomyosis (9.9 cells / ×

200), eutopic endometrium in the secretory phase (4.17 cells / × 200) ,

peritoneal / pelvic endometriosis (3.63 cells / × 200), proliferative phase

eutopic endometrium (2.33 cells / × 200) and ovarian endometriosis (1.99

cells / × 200).

The average CD68 + macrophage cell density varied as follows:

adenomyosis (65.6 cells / × 200), abdominal wall endometriosis (57.66 cells /

× 200), eutopic endometrium in secretory phase (12.11 cells / × 200) ,

peritoneal / pelvic endometriosis (11.96 cells / × 200), ovarian endometriosis

(11.94 cells / × 200), and proliferative phase eutopic endometrium (10.01

cells / × 200).

The average mast cell type Triptase + density varied as follows:

adenomyosis (19.34 cells / × 200), endometriosis of the abdominal wall

(18.81 cells / × 200), peritoneal / pelvic endometriosis (4.77 cells / × 200),

ovarian endometriosis (3.51 cells / × 200), secretory phase eutopic

endometrium (3.45 cells / × 200) and proliferative phase eutopic

endometrium (2.59 cells / × 200). Also, the mean cell density of CD79α +

23

lymphocyte type B varied as follows: abdominal wall endometriosis (29.05

cells / × 200), adenomyosis (12.45 cells / × 200), ovarian endometriosis

(11.82 cells / × 200) ), peritoneal / pelvic endometriosis (9.96 cells / × 200),

eutopic endometrium in secretory phase (8.81 cells / × 200) and eutopic

endometrium in proliferative phase (8.41 cells / × 200).

We compared the averages obtained for each category and acquired an

overall result. There is a statistically significant difference, depending on the

location of the endometriosis, for the values: CD34 - F (5,179) = 596,510, p

<0.001; CD3 - F (5,179) = 429,196, p <0.001; CD20 - F (5,179) = 940.025, p

<0.001; CD68 - F (5,179) = 758,489, p <0.001; Triptase - F (5,179) =

808,694, p <0.001; CD79α - F (5,179) = 448,372, p <0.001.

Conclusions: Immunohistochemical tests conducted for the

identification of glandular epithelium, hormonal receptors, tissue

proliferation, but also for the identification of tumor proteins, vascularization

and inflammatory cells, were the basis for the positive diagnosing of

endometriosis with different locations and to highlight the possibility of

preneoplastic transformation.

The histopathological examination has a certain diagnostic value and

dictates the choice of therapeutic behavior.

The higher the production of sex hormones, the higher the number of

blood vessels and positive immunohistochemical reactions for the

inflammatory cell line: CD3 +, CD20 +, CD68 +, Tryptase, CD79α +, the

higher the endometrial cell dissemination rate.

Disseminated cells and inflammatory response cause the most common

symptoms of endometriosis: chronic pelvic pain and infertility.

The mediators secreted by the inflammatory cells cause important

changes of the histoarhitecture of the microenvironment adjacent to

endometriosis / adenomyosis outbreaks, but also to ectopically implanted

endometrial cells, which may lead to the appearance of cellular atypia.

24

CHAPTER VIII: Final conclusions

Endometriosis / adenomyosis are benign pathologies of genital origin,

which occur more frequently in women of childbearing (reproductive) age.

The extensive study to identify the ectopic endometrial structures and

their importance in the prognosis of the disease were the targets pursued

within this thesis.

Under the influence of certain stimuli, of the anatomy of the female

reproductive organs and of the inflammatory processes, viable endometrial

cells can migrate and can be implanted, ectopically, in the structure of the

different organs.

The most common symptoms are chronic pelvic pain, vaginal bleeding

and infertility.

The correct and rapid medical investigations, together with the

application of the appropriate treatment substantially reduces the risk of

infertility and reduces the symptomatology.

The current study methods are of major importance in the choice of

therapeutic possibilities and in the prognosis of the disease.

A multitude of inflammatory, hormonal, mechanical factors are

involved in the progression of this pathology.

Through the clinical study we have observed that the age of the patients

can fluctuate, that the body mass index can influence the appearance of

endometriosis / adenomyosis, thus, the normoponderal or underweight

women are more predisposed to the development of this pathology, compared

with the overweight ones, due to the decrease of the amount of the sex

hormones. and most cases of primary infertility were observed in women

with ovarian endometriosis.

With regard to laboratory tests, serum ESR, CA-125 may be elevated,

and secondary anemia may occur in patients with heavy bleeding.

25

From a microscopic point of view, we observed the existence of

glandular epithelium and periglandular stroma having a similar appearance to

the normal endometrial tissue, in the structure of the different organs.

Using the immunohistochemical study with the help of anti-CK7 /

CK20 antibodies we have shown that ectopic tissue has epithelial-

endometrial origin and that these structures are not possible metastases from

gastrointestinal tumors.

Marking with anti-ER and anti-PR antibodies has shown us that the

areas studied are endometriosis / adenomyosis foci. Estrogen maintains the

survival and dissemination of endometriotic cells, and the presence of ER in

the structure of endometriosis / adenomyosis outbreaks shows an increased

tissue responsivity to this hormone. Positive immunohistochemical staining

with anti-ER and anti-PR antibodies may guide the physician in applying

hormonal treatment to patients diagnosed with this pathology.

The presence of cell apoptosis inhibitory genes (BCL-2, PTEN)

increases the rate of cell disruption (highlighted by the anti-Ki67 antibody),

the determination of intracellular tumor protein 53 (evidenced by the anti-p53

antibody) and the strong immune response may influence the malignant

transformation of endometriosis / adenomyosis outbreaks.

The periglandular inflammatory process highlighted with the help of

anti-CD3 / CD20 / CD68 / Triptase / CD79α antibodies demonstrates its

involvement in the evolution and possibility of preneoplastic transformation

through secreted mediators, which produce important changes in the histo-

architecture of the microenvironment adjacent to the endometriosis /

adenomyosis foci and ectopically implanted endometrial cells, which can

lead to the appearance of cellular atypia.

The intense periglandular vascularization is involved in the processes

of cell growth and proliferation, supports the hypothesis suggested by

hematogenous dissemination, but may also influence the evolution of

migrated cells.

26

The effective treatment of endometriosis remains the surgical

intervention, thus suppressing the pain cause and preventing the preneoplastic

/ malignant transformation of the endometriosis / adenomyosis outbreaks.

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